Epoxyeicosatrienoic acids (EETs) form adducts with DNA in vitro

• Published in: Prostaglandins & other lipid mediators Vol. 123; pp. 63 - 67
• Main Authors: Funk, Dorothee J., Sorg, Bernd L., Kopka, Klaus, Schmeiser, Heinz H.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2016
• Subjects: 32P-postlabelling; 8,11,14-Eicosatrienoic Acid - analogs & derivatives; 8,11,14-Eicosatrienoic Acid - chemistry; Animals; Arachidonic acid; Cattle; Cytochrome P450; Deoxyadenine Nucleotides - chemistry; Deoxyguanine Nucleotides - chemistry; DNA - chemistry; DNA Adducts - chemical synthesis; DNA damage; EETs; Hydrogen-Ion Concentration; Kinetics; Lipid mediator; Phosphorus Radioisotopes - chemistry; Solutions; Stereoisomerism; Arachidonic acid; Lipid mediator; DNA damage; Cytochrome P450; 32P-postlabelling; EETs; P-postlabelling
• ISSN: 1098-8823
• DOI: 10.1016/j.prostaglandins.2016.04.006

•Epoxyeicosatrienoic acids (EETs) react with DNA in vitro at pH 4.•EET-DNA adducts are detected by the 32P-postlabelling method.•Under physiological conditions (pH 7.4) only ±11,12-EET formed adducts with DNA.•EETs might be endogenous genotoxic compounds. Epoxyeicosatrienoic acids (EETs) are potent lipid mediators formed by cytochrome P450 epoxygenases from arachidonic acid. They consist of four regioisomers of cis-epoxyeicosatrienoic acids: 5,6-, 8,9-, 11,12- and 14,15-EET. Here we investigated whether these triene epoxides are electrophilic enough to form covalent adducts with DNA in vitro. Using the thin-layer chromatography (TLC) 32P-postlabelling method for adduct detection we studied the reaction of individual deoxynucleoside 3′-monophosphates and calf thymus DNA with the four racemic EETs. Under physiological conditions (pH 7.4) only ±11,12-EET11,12-EET formed adducts with DNA in a dose dependent manner detectable by the 32P-postlabelling method. However, when pre-incubated at pH 4 all four racemic EETs were capable to bind to DNA forming several adducts. Under these conditions highest DNA adduct levels were found with ±11,12-EET followed by ±5,6-EET, ±8,9-EET, and ±14,15-EET, all of them two orders of magnitude higher (between 3 and 1 adducts per 105 normal nucleotides) than those obtained with ±11,12-EET at pH 7.4. Similar DNA adduct patterns consisting of up to seven spots were observed with all four racemic EETs the most abundant adducts being derived from the reaction with deoxyguanosine and deoxyadenosine. In summary, when analysed by the 32P-postlabelling method all four racemic EETs formed multiple DNA adducts after activation by acidic pH, only ±11,12-EET produced DNA adducts in aqueous solution at neutral pH. Therefore, we conclude from our in vitro studies that EETs might be endogenous genotoxic compounds.